Metabolic and Developmental Effects Resulting from Deletion of the citA Gene Encoding Citrate Synthase in Aspergillus nidulans

Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.There has been increased interest in primary carbon metabolism in fungi in recent years. There are two main reasons for this. As fungal pathogens establish infection they must adapt their utilization of carbon sources to the substrates present in the new environment of the host cells (reviewed in reference 6). With many of the fungal genomes available, the number of genes encoding enzymes and transporters potentially involved in central metabolism has become apparent and is greater than might have been anticipated (for example, see reference 16). Deciphering this complexity requires not only genome-wide studies but also detailed studies of individual genes encoding these proteins in order to determine their regulation and the cellular localization of the proteins, as well as their roles in metabolism and development. Here we report molecular genetic analysis of the citA gene encoding citrate synthase (EC 4.1.3.7), a central enzyme of carbon metabolism, in the filamentous ascomycete Aspergillus nidulans.Citrate synthase is required for the formation of citrate from acetyl-coenzyme A (CoA) and oxaloacetate in the tricarboxylic acid (TCA) cycle and is therefore necessary for respiratory growth as well as for the generation of intermediates for biosynthetic reactions. Together with aconitase, malate dehydrogenase, isocitrate lyase, and malate synthase, it is also an essential enzyme in the glyoxylate cycle, which is necessary for growth on carbon sources such as acetate, ethanol, and fatty acids which are catabolized via acetyl-CoA (reviewed in reference 26).In Saccharomyces cerevisiae the mitochondrial Cit1 is the major citrate synthase of the TCA cycle. An additional enzyme, Cit2, is peroxisomally localized via a C-terminal peroxisomal targeting sequence (PTS1) (29). In response to mitochondrial dysfunction CIT2 is upregulated via the retrograde response mediated by RTG1, -2, and -3, while mitochondrial respiratory deficiency results in RTG-dependent expression of CIT1 as well as that of aconitase (ACO1) and isocitrate dehydrogenase (IDH1 and IDH2), all enzymes necessary for 2-oxoglutarate formation and hence the synthesis of glutamate required for amino acid biosynthesis (9, 15, 30). In addition a third gene, CIT3, encodes a mitochondrial enzyme with citrate synthase activity. This enzyme has greater activity with propionyl-CoA, forming methylcitrate, and is necessary for the mitochondrial methylcitrate cycle involved in the metabolism of propionate (24). Cit2 has also been proposed to have methylcitrate synthase activity (17).In S. cerevisiae Cit2 also plays a role in the transfer of acetyl-CoA generated in peroxisomes by β-oxidation of fatty acids or by ethanol and acetate metabolism in the cytoplasm to the mitochondria for metabolism via the TCA cycle. There are two alternative pathways: transfer as acetyl-carnitine formed by the peroxisomal/mitochondrial carnitine acetyltransferase Cat2, together with the cytoplasmic Yat1 and Yat2 carnitine acetyltransferases, or transfer via citrate formed by Cit2 (45, 51, 52). Only disruption of both pathways (e.g., by deletion of CAT2 and CIT2) results in a growth defect on fatty acids. The fact that deletion of CIT2 is not essential for utilization of carbon sources metabolized via acetyl-CoA indicates that mitochondrial citrate synthase activity can replace the peroxisomal activity in the glyoxylate cycle. In contrast, in the pathogenic yeast Candida albicans, there is a single gene for citrate synthase and it is mitochondrial, and acetyl-CoA transport to mitochondria is solely dependent on the carnitine pathway (43, 57). In the plant Arabidopsis thaliana, there are five genes encoding citrate synthase enzymes. Two are peroxisomal (CSY2 and CSY3) and required for fatty acid respiration and seed germination, indicating that carnitine acetyltransferases are not required for shuttling acetyl units to the mitochondria (37).The filamentous ascomycete Aspergillus nidulans has both citrate synthase-encoding and methylcitrate synthase-encoding genes, citA and mcsA, respectively (8, 36). In both A. nidulans and Aspergillus fumigatus it has been shown that McsA is mitochondrial and has both methylcitrate and citrate synthase activities and is required for propionyl-CoA metabolism (8, 22, 31). Cell fractionation studies have shown that citrate synthase activity colocalizes with the mitochondrial fraction (35), and an N-terminal mitochondrial targeting sequence is predicted by the gene sequence (36). However, CitA has a putative C-terminal peroxisomal targeting sequence (PTS1 AKL), and genes in some filamentous ascomycetes also have potential PTS1 sequences (see below). The role of peroxisomal citrate synthase activity is not at all clear. The acuJ-encoded peroxisomal/mitochondrial carnitine acetyltransferase is required for growth on both fatty acids and acetate, while the facC-encoded cytoplasmic enzyme is required for growth on acetate (1, 20, 42). Therefore, like C. albicans, the carnitine shuttle is absolutely required for acetyl-CoA intracellular transport.Because of our interest in the role of peroxisomes in fatty acid and acetate metabolism in A. nidulans (21), we have investigated phenotypes resulting from deletion of the citA gene. Our results indicate that loss of CreA-mediated carbon repression allows expression of mcsA, resulting in the restoration of sufficient citrate synthase activity to suppress growth and developmental defects resulting from citAΔ. We have also investigated the role of peroxisomal localization of CitA and found this is at most extremely low and does not play a major role.